An understanding of the structure and working of the Na/K-pump is important for this protein generates the trans-membrane gradients of Na and K which maintain electrical excitability, volume control, secretory activities etc and may also be involvement in pathophysiology of diseases such as essential hypertension. Despite a mass of knowledge on structure-function relations, the central question for the Na/K-pump (and all other pumps)-the manner in which the free energy of hydrolysis of ATP is transduced into active cation movements - remains unanswered. For the Na/K-pump there is some knowledge as to residues in the cytoplasmic domain of the alpha chain with which ATP interacts. Little or nothing is known about residues that ligate the cations or the location of the cation sites. This is a major challenge since interactions between the cation and ATP sites are the essence of energy transduction. This application proposes to investigate the structure and working of the cation binding sites of renal cell membrane Na/K-ATPase, selecting two different but complementary areas of study. (A) We shall attempt to identify polypeptide segments involved in cation binding and movements, by utilising chemical modification by dicyclohexyl carbodiimide (DCCD) combined with selective proteolytic digestion. We have proposed previously that two K or two Na ions are bound by the same two carboxyl residues, while the third Na is complexed with neutral oxygen ligands. Following a dramatic recent finding we propose that cation binding residues reside on a19 KD tryptic fragment of the alpha chain, containing two putative C- terminal trans-membrane segments. The membrane-embedded 19KD fragment is selectively labelled by DCCD; thus identifying the relevant carboxyls now seems feasible. In the membrane it is likely that several trans-membrane segments form the ion binding cage. An attempt to identify such segments will combine studies of photolabelling with hydrophobic probes, radiation inactivation, and solubilisation and purification of proteolysed membranes containing tryptic fragments but still able to occlude cations. (B) Studies on electrogenicity and voltage-sensitivity of the renal Na/K-ATPase reconstituted into proteoliposomes provide insight into charge movements in cation sites. These have interesting structural implications. For example, observations of voltage effects on active transport suggested that the sites contain two mobile negative charges, consistent with the chemical modification. We now intend to explore the possibility of voltage effects on cation binding affinities, for if found these may suggest the existence of 'ion wells' at a depth within the protein. Studies of electrogenic potentials using the dye Oxonol VI have suggested that many conditions coupling ratios of the pump are constant (e.g. the classical 3Na/2K) but at low concentrations of transported ions they may fall. We intend to investigate and test this surprising hypothesis, for if verified it will have important implications for the flexibility of the interaction between the cation and ATP sites.